Homocysteine (Hcy), a thiol-containing amino acid, is a metabolic intermediate of both methionine (Met) and cysteine (Cys) production. The exclusive source of Hcy in mammals is a three-step conversion of the essential dietary amino acid Met in the "active methyl cycle". (Scheme 1).
Hcy may be removed from the active methyl cycle to combine with serine to form Cys via a cystathionine intermediate, a reaction which is catalyzed by cystathionine synthase (CS). Alternatively, Hcy may participate in the active methyl cycle by being methylated to Met, a reaction which is catalyzed by methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase). The major metabolic pathway for the methylation of Hcy to Met is dependent on the cofactor activity of folate and vitamin B.sub.12. Not surprisingly, elevated levels of serum Hcy have been associated with insufficient intake of vitamin B.sub.12 or folate, or a deficiency in the ability to properly utilize these vitamins. ##STR1##
Decreased levels of folate and Vitamin B.sub.12, and thus a significantly higher Hcy concentration, have been observed in the amniotic fluid of pregnancies where the fetus was affected with a neural tube defect. Moderately elevated levels of Hcy usually can be brought into balance by administering folate, a treatment for which there are few adverse side-effects.
Elevated levels of Hcy are present in the serum and urine of patients with cystathionine synthetase deficiency who cannot convert Hcy to cystathionine, and in the serum and urine of patients with defects involving methionine synthetase who cannot convert Hcy to Met (Mudd, S H, in The Metabolic Basis of Inherited Disease (Senver, C G, ed.) McGraw Hill, N.Y., pp.693-734 (6.sup.th ed. 1989)). These genetic predispositions are the most common causes of homocysteinuria (build-up of Hcy in urine) in otherwise healthy patients. The clinical features of this disease are early, life threatening thromboembolism, mental retardation, and other tissue abnormalities.
Hcy in high concentrations is generally considered to be atherogenic and thrombogenic. In the last decade, over twenty case-controlled studies have consistently shown that plasma Hcy concentration is very frequently increased in patients with vascular disease (Ueland et al., in Atherosclerotic Cardiovascular Disease, Hemostasis and Endothelial Function (Francis, R B. Jr., ed.) NY, pp.183-236 (1992)). An excellent overview of the causes of homocysteinuria as well as an update on the current methods of clinical analysis can be found in Ueland et al. (1993) Clin. Chem. 39:1764-1779. A study by Graham et al. (1997) JAMA 277:1775-1781, confirms that an elevated plasma total Hcy level is established as a strong and independent factor associated with all categories of atherosclerotic disease in both men and women.
Thus, plasma and serum Hcy are established as indicators of several common disease states. Therefore, the clinical importance of Hcy has lead to an increased interest in developing an accurate assay for measuring Hcy in biological fluids.
Hcy exists in human plasma as various mixed disulfides. Normally, a major fraction of Hcy (approximately 70%) is protein bound via a disulfide bond to circulating proteins such as albumin. The remaining "free" Hcy (approximately 30%) is in the form of Hcy (reduced) or as mixed disulfides with other thiols such as Cys. The sum of these Hcy species present in plasma (protein-bound, free-disulfide and free-reduced) is referred to as the "total Hcy." Measurement of total Hcy in biological fluids preferably involves a pretreatment step to form the free reduced-form Hcy. However, measurement of free Hcy may be done without pretreatment, if desired. There are several techniques to quantitate total homocysteine (Hcy) as well as distinguish between the free (reduced and disulfide) and protein-bound (primarily albumin) forms.
The detection and/or separation of Hcy in biological samples by current methods is difficult due to the presence of multiple sulfhydryl species present in the sample. During the last two decades, assays for Hcy and Cys have mainly involved high performance liquid chromatographic (HPLC) methods. This analytical method discriminates between Hcy and Cys by differential adsorption and elution of the compounds on a chromatographic support. Many of these methods involve pre- or postcolumn derivatization of plasma sulfhydryls, for example, with ninhydrin (Clarke et al. (1991) N. Engl. J. Med. 324:1149-1155; Kang et al. (1986) J. Clin. Invest. 77:1482-1486; Andersson et al. (1992) Eur. J. Clin. Invest. 22:79-87), monobromobimane (Fiskerstrand et al. (1993) Clin. Chem. 39:263-271; Mansoor et al. (1992) Anal. Biochem. 200:218-229; Fahey et al. (1981) Anal. Biochem. 111:357-365; Velury and Howell (1988) J. Chromatography 424:141-146; Jacobsen et al. (1989) Anal. Biochem. 178:208-214; Refsum et al. (1989) Clin. Chem. 35:1921-1927), adenosine (Refsum et al. (1985) Clin. Chem. 31:624-628), N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (Stabler et al. (1987) Anal. Biochem. 162:185-196), halogenated sulfonyl benzofurazans (Araki and Sako (1987) J. Chromatography 422:43-52; Ubbink et al. (1991) J. Chromatography 565:441-446; Vester et al. (1991) Eur. J. Clin. Chem. Clin. Biochem. 29:549-554), o-phthaldialdehyde (Fermo et al. (1992) J. Chromatography 593:171-176), 4,4'-dithiodipyridine (Andersson et al. (1993) Clin. Chem. 39:1590-1597), 5,5'-dithiobis(2-nitrobenzoic acid) (Kuwata et al. (1982) Anal. Chem. 54:1082-1087; Reeve et al. (1980) J. Chromatography 194:424-428; Konouro et al. (1985) J. Chromatography 338:209-212; Studebaker et al. (1978) Anal. Chem. 50:1500-1503), or with electrochemical detection (Swift et al. (1986) Nutr. Reports, Int. 34:1-4; Manilow et al. (1989) Circulation 79:1180-1188; Smolin et al. (1982) J. Nutr. 112:1264-1272). Some of these methods, namely, electrochemical (Manilow) or derivatization with ninhydrin (Clark, Andersson, Kang), monobromobimane (Refsum) or N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (Stabler) have been used to measure Hcy in larger amounts of biological samples. Only monobromobimane (Mansoor) and halogenated sulfonyl benzofurazans (Araki) were able to detect other sulfhydryls in plasma. Recently (Mansoor, Andersson) methods have been described that measured total, free (non-protein-bound) and reduced Hcy in plasma.
In an example of Hcy analysis by HPLC which requires prior derivatization with fluorescent labels, such as bromobimane, the bromomethyl group reacts with the free thiol of Hcy, thus forming a thioether. A problem with this method, however, is that the bromobimane reagent also reacts with all other free thiols in solution. Therefore, chromatographic separation of the various derivatized sulfur-containing species is necessary. Thus, chromatographic methods have the disadvantages of being slow, labor intensive and expensive.
An enzymatic method for a Hcy assay is described by Sundrehagen et al., WO93/15220, where Hcy is assayed indirectly by measuring the product concentration following the enzyme catalyzed conversion of Hcy to S-adenosyl homocysteine.
Methods of analyzing sulfhydryl amino acids by gas chromatograph-mass spectrometer (GC-MS) means are known. Allen et al. (U.S. Pat. No. 4,940,658), describe analysis of, for example, Hcy by GC-MS using labeled Hcy as an internal reference standard.
Van Atta et al. (U.S. Pat. No. 5,478,729), describe a method of detecting Hcy in the presence of Cys comprising chemically modifying Hcy and Cys and then immunochemically detecting the modified Hcy by means of an antibody which specifically binds to the modified Hcy but not to the modified Cys.
Stern et al. (J. Biochem. and Biophys. Methods 7:83-88 (1982)) describe a method of purifying Met-free [.sup.35 S]Hcy thiolactone following its synthesis. In this method, [.sup.35 S]Hcy thiolactone is synthesized by treating [.sup.35 S]Met under harsh conditions (refluxing the Met in hypophosphorous acid and hydriodic acid for 22 hours), followed by chromatographic purification of the Hcy thiolactone on an alumina column. Met and any unreacted Hcy are retained by the alumina through its carboxyl group and are not eluted. The yield of the Hcy thiolactone by this procedure was only 5-10%.
A method for converting Hcy to Hcy thiolactone and an assay for determining this conversion spectrophotometrically at 240 nm was described by Racker (J. Biol. Chem. 217:867-874 (1955)). However, it was not recognized by Racker that this method could be used to selectively separate the thiolactone from free thiol-containing compounds. Furthermore, as evidenced by the cumbersome chromatographic methods currently used to assay for Hcy, the conversion of Hcy to Hcy thiolactone has clearly not been recognized as a useful tool for incorporation in a Hcy assay.
A need exists for an improved assay for Hcy which is simple, specific, quick to perform, readily adapted for use in clinical laboratories, and which avoids the need for costly and time consuming chromatographic separation.